Nitric oxide is a potential regulator of mitochondrial biogenesis. Therefore, we investigated if mice deficient in endothelial nitric oxide synthase (eNOS−/−) or neuronal NOS (nNOS−/−) have attenuated activation of skeletal muscle mitochondrial biogenesis in response to exercise. eNOS−/−, nNOS−/− and C57Bl/6 (CON) mice (16.3 ± 0.2 weeks old) either remained in their cages (basal) or ran on a treadmill (16 m min−1, 5% grade) for 60 min (n= 8 per group) and were killed 6 h after exercise. Other eNOS−/−, nNOS−/− and CON mice exercise trained for 9 days (60 min per day) and were killed 24 h after the last bout of exercise training. eNOS−/− mice had significantly higher nNOS protein and nNOS−/− mice had significantly higher eNOS protein in the EDL, but not the soleus. The basal mitochondrial biogenesis markers NRF1, NRF2α and mtTFA mRNA were significantly (P< 0.05) higher in the soleus and EDL of nNOS−/− mice whilst basal citrate synthase activity was higher in the soleus and basal PGC-1α mRNA higher in the EDL. Also, eNOS−/− mice had significantly higher basal citrate synthase activity in the soleus but not the EDL. Acute exercise increased (P< 0.05) PGC-1α mRNA in soleus and EDL and NRF2α mRNA in the EDL to a similar extent in all genotypes. In addition, short-term exercise training significantly increased cytochrome c protein in all genotypes (P< 0.05) in the EDL. In conclusion, eNOS and nNOS are differentially involved in the basal regulation of mitochondrial biogenesis in skeletal muscle but are not critical for exercise-induced increases in mitochondrial biogenesis in skeletal muscle.